1. Field of the Invention
The present invention relates to a method for separating a material that has affinity to an antibody moiety of a protein-antibody complex which has modified partition characteristics, more precisely an affinity separation method to separate a material specifically binds to an antibody moiety of the complex, in which a protein changes the partition characteristics of the protein-antibody complex.
2. Description of the Related Art
Separation of biomolecules is a very important, essential technique in the production of biological molecules including pharmaceutics, cell therapy industries, which are growing everyday. However, biomolecules are fragile comparing with chemical compounds, and have disadvantage of complicated purification process because of numbers of similar materials having similar physical and chemical characteristics in the solution. The separation of biomolecules via aqueous two-phase extraction system favors maintaining the stability of biomolecules, since this technique does not require an organic solvent, and makes the complicated purification process simple. Aqueous two-phase extraction system is composed of two different aqueous solutions containing high-molecular substances or high molecular substance and salt together at a required content, and at this time, two phases are formed in the system which are immiscible together because of electrostatic repulsion force between the substances.
The principal of the separation using aqueous two-phase extraction system is as follows; when high molecular weight substance such as polyethylene glycol and salt such as phosphate are mixed at a certain ratio, it is divided into two phases, the upper polyethylene glycol phase and the lower phosphate phase, owing to the difference of density and the molecular level repulsion between polyethylene glycol and salt. At this time, when the target materials for separation are added, these materials are crowded in one of the two phases according to their preference, resulted from the principle that each phase has different physical, chemical characteristics according to its components and compositions.
Mainly used polymer for composing aqueous two-phase extraction system is exemplified by polyethylene glycol, polyvinyl alcohol, dextran and oligosaccharide. The salt used for forming aqueous two-phase extraction system is exemplified by potassium phosphate, sodium phosphate, ammonium sulfate and magnesium sulfate. Aqueous two-phase extraction system contains water at least 65-80%, by which stability of biomolecules can be maintained. Such aqueous two-phase extraction system can be formed by various materials, which favors separation of biomolecules.
Up to date, it has been attempted in aqueous two-phase extraction system to change concentrations of materials forming the system and molecular weights of high molecular substances according to a target material for separation. In particular, various salts have been tried because salts affect partition characteristics significantly by changing surface properties of a target material or hydrophobicity of aqueous two-phase extraction system. However, the addition of salts increases osmotic pressure in solution, which might reduce stability of biomolecules and decreases selectivity because it affects not only the target material but also partition coefficients of other materials. So, it is very difficult to determine the optimum kind and concentration of such salt.
To overcome the above problem, it has been tried to use biological or chemical affinity in aqueous two-phase extraction system. The material used for enhancing affinity in aqueous two-phase extraction system is exemplified by antibodies, DNA, metal ions and surfactants.
In relation to this invention, there was an example of affinity separation using an antibody in aqueous two-phase extraction system, in which a temperature sensitive polymer was conjugated to an antibody as a ligand, to separate animal cells at a certain temperature (A. Kumar et al., Biotechnol. Bioeng., 75, 570-580, 2005). But, to increase separation efficiency, the temperature had to be maintained unchanged, otherwise biomolecules would be denatured. Besides, the toxicity of the polymer used as a ligand should be considered.
Another example of affinity separation is to use PEGylated molecules prepared by conjugating an antibody to PEG (polyethylene glycol) (Kim A. Sharp et al. Anal. Biochem. 154, 110-117, 1985). However, this method has disadvantage of high price of modified PEG and the success of PEGylation might be vulnerable according to the modified PEG and antibody types.
Thus, in order to separate a target material selectively from impurities without damaging stability of biomolecules, the present inventors choose protein and aqueous two-phase extraction system which are capable of distributing target materials on the opposite phase from the one where the antibodies are distributed. Then, the present inventors completed this invention by confirming that the target materials mixed in impurities can be recovered by re-distributing to opposite phase.